Method for determining cell cycle position

ABSTRACT

The invention provides a novel, non-destructive and dynamic process for determining the cell cycle position of living cells. The invention also provides DNA constructs, and cell lines containing such constructs, that exhibit activation and deactivation of a detectable reporter molecule in a cell cycle specific manner. The invention thus allows greater precision in determining cell cycle phase status than existing techniques and further provides a method for continuous monitoring of cell cycle progression in individual cells.

TECHNICAL FIELD

[0001] The present invention relates to a novel, non-destructive anddynamic process for determining the cell cycle position of living cells.

BACKGROUND TO THE INVENTION

[0002] Eukaryotic cell division proceeds through a highly regulated cellcycle comprising consecutive phases termed G1, S, G2 and M. Disruptionof the cell cycle or cell cycle control can result in cellularabnormalities or disease states such as cancer which arise from multiplegenetic changes that transform growth-limited cells into highly invasivecells that are unresponsive to normal control of growth. Transition ofnormal cells into cancer cells can arise arise though loss of correctfunction in DNA replication and DNA repair mechanisms. All dividingcells are subject to a number of control mechanisms, known as cell-cyclecheckpoints, which maintain genomic integrity by arresting or inducingdestruction of aberrant cells. Investigation of cell cycle progressionand control is consequently of significant interest in designinganticancer drugs. (Flatt, P. M. and Pietenpol, J. A. Drug Metab. Rev.,(2000), 32(3-4), 283-305; Buolamwini, J. K. Current PharmaceuticalDesign, (2000), 6, 379-392).

[0003] Accurate determination of cell cycle status is a key requirementfor investigating cellular processes that affect the cell cycle or aredependent on cell cycle position. Such measurements are particularlyvital in drug screening applications where:

[0004] i) substances which directly or indirectly modify cell cycleprogression are desired, for example, for investigation as potentialanti-cancer treatments;

[0005] ii) drug candidates are to be checked for unwanted effects oncell cycle progression; and/or

[0006] iii) it is suspected that an agent is active or inactive towardscells in a particular phase of the cell cycle.

[0007] Traditionally, cell cycle status for cell populations has beendetermined by flow cytometry using fluorescent dyes which stain the DNAcontent of cell nuclei (Barlogie, B. et al, Cancer Res., (1983), 43(9),3982-97). Flow cytometry yields quantitative information on the DNAcontent of cells and hence allows determination of the relative numbersof cells in the G1, S and G2+M phases of the cell cycle. However, thisanalysis is a destructive non-dynamic process and requires serialsampling of a population to determine cell cycle status with time.Furthermore, standard flow cytometry techniques examine the total cellpopulation in the sample and yield limited data on individual cells,which precludes study of cell cycle status of different cell types thatmay be present within the sample under analysis.

[0008] EP 798386 describes a method for the analysis of the cell cycleof cell sub-populations present in heterogeneous cell samples. Thismethod uses sequential incubation of the sample with fluorescentlylabelled monoclonal antibodies to identify specific cell types and afluorochrome that specifically binds to nucleic acids. This permitsdetermination of the cell cycle distribution of sub-populations of cellspresent in the sample. However, as this method utilises flow cytometry,it still yields only non-dynamic data and requires serial measurementsto be performed on separate samples of cells to determine variations inthe cell cycle status of a cell population with time following exposureto an agent under investigation for effects on cell cycle progression.

[0009] A further disadvantage of flow cytometry techniques relates tothe indirect, and inferred assignment of cell cycle position of cellsbased on DNA content. Since the DNA content of cell nuclei variesthrough the cell cycle in a reasonably predictable fashion, ie. cells inG2 or M have twice the DNA content of cells in G1, and cells undergoingDNA synthesis in S phase have an intermediate amount of DNA, it ispossible to monitor the relative distribution of cells between differentphases of the cell cycle. However, the technique does not allowprecision in determining the cell cycle position of any individual celldue to ambiguity in assigning cells to G2 or M phases and to furtherimprecision arising from inherent variation in DNA content from cell tocell within a population which can preclude precise discriminationbetween cells which are close to the boundary between adjacent phases ofthe cell cycle. Additionally, variations in DNA content and DNA stainingbetween different cell types from different tissues or organisms requirethat the technique is optimised for each cell type, and can complicatedirect comparisons of data between cell types or between experiments(Herman, Cancer (1992), 69(6), 1553-1556). Flow cytometry is thereforesuitable for examining the overall cell cycle distribution of cellswithin a population, but cannot be used to monitor the precise cellcycle status of an individual cell over time.

[0010] Cell cycle progression is tightly regulated by defined temporaland spatial expression, localisation and destruction of a number of cellcycle regulators which exhibit highly dynamic behaviour during the cellcycle (Pines, J., Nature Cell Biology, (1999), 1, E73-E79). For example,at specific cell cycle stages some proteins translocate from the nucleusto the cytoplasm, or vice versa, and some are rapidly degraded. Fordetails of known cell cycle control components and interactions, seeKohn, Molecular Biology of the Cell (1999), 10, 2703-2734.

[0011] One of the most extensively characterised cell cycle regulatorsin human cells is cyclin B1, temporal and spatial expression anddestruction of which controls cell transition from G2 to M and its exitfrom M. Cyclin B1 expression is driven by a cell cycle phase specificpromoter which initiates expression at the end of S phase and peaksduring G2. Once expressed, this protein constantly shuttles between thenucleus and the cytoplasm during the G2 phase, but it is primarilycytoplasmic because the rate of its export is much greater than itsimport. At the start of mitosis, cyclin B1 rapidly translocates into thenucleus, when its rate of import substantially increases, and its exportdecreases, in a phosphorylation dependent manner (FIG. 1). Thus, thelocalisation of cyclin B1 in the cell can be used to mark the transitionfrom G2 phase to mitosis. Once a cell reaches metaphase, or, moreaccurately, when the spindle assembly checkpoint is satisfied, cyclin B1is very rapidly degraded. Cyclin B1 destruction continues throughout thefollowing G1 phase but stops once cells begin DNA replication. Theseevents have been visualised in real time by micro-injection offluorescently labelled cyclin B1 into living cells (Clute and Pines,Nature Cell Biology, (1999), 1, 82-87).

[0012] The controlling elements which regulate temporal expression anddestruction have been elucidated in a number of studies. Biosynthesis ofcyclin B1 has been shown to be controlled at the level of transcriptionby a promoter sequence that confines expression to the late S and G2phases of the cell cycle (Piaggio et al, Exp. Cell. Research, (1995),216, 396-402; Cogswell et al, Mol.Cell. Biology, (1995), 15, 2782-2790).Destruction of cyclin B1 at the appropriate time in M phase has beenshown to be controlled by a 9 amino acid sequence, termed thedestruction box (D-box) which targets the protein for proteolysis viaubiquitinylation. Expression of a Drosophila cyclin B-GFP fusion proteindriven by a constitutive polyubiquitin promoter (Huang and Raff, EMBOJournal, (1999), 18(8), 2184-2195) has shown that fluorescently-taggedcyclin B mimics the behaviour of endogenous cyclin B in being degradedat the end of metaphase. Studies (Hagting et al, Current Biology,(1999), 9, 680-689) using human cyclin B1-GFP have shown that temporalchanges in cytoplasmic and nuclear localisation of cyclin B1 with cellcycle progression is dependent on a nuclear export signal (NES),phosphorylation of which leads to nuclear import.

[0013] Other cell cycle checkpoints are similarly regulated by temporaland spatial control mechanisms and many of the components andinterrelationships have been elucidated (Pines, J., Nature Cell Biology,(1999), 1, E73-E79).

[0014] A number of methods have been described which make use of certaincomponents of the cell cycle control mechanisms to provide procedureswhich analyse or exploit cell proliferation status.

[0015] WO 00/29602 describes use of a cyclin A promoter to driveexpression of GFP as a selectable marker for dividing transgenic stemcells to allow dividing cells to be isolated from a background ofnon-dividing cells by fluorescence activated cell sorting. While thismethod allows identification and selection of cells which haveprogressed past a certain stage in the cell cycle, it does not yieldinformation on the cell cycle status of any given cell, other thanhistorical information that the cell has or has not passed through theG2 phase of the cell cycle at some time in the past.

[0016] U.S. Pat. No. 6,048,693 describes a method for screening forcompounds affecting cell cycle regulatory proteins, wherein expressionof a reporter gene is linked to control elements which are acted on bycyclins or other cell cycle control proteins. In this method, temporalexpression of a reporter gene product is driven in a cell cycle specificfashion and compounds acting on one or more cell cycle controlcomponents may increase or decrease expression levels. Since the assaysystem contains no elements which provide for the destruction of thereporter gene product nor for destruction of any signal arising from thereporter gene, the method cannot yield information on the cell cycleposition of any cells in the assay and reports only on generalperturbations of cell cycle control mechanisms.

[0017] U.S. Pat. No. 5,849,508 and U.S. Pat. No. 6,103,887 describemethods for determining the proliferative status of cells by use ofantibodies which bind to cyclin A. These methods provide means fordetermining the percentage of proliferating cells in a test populationrelative to a control population.

[0018] U.S. Pat. No. 6,159,691 relates to a method for assaying forputative regulators of cell cycle progression. In this method, nuclearlocalisation signals (NLS) derived from the cell cycle phase specifictranscription factors DP-3 and E2F-1 are used to assay the activity ofcompounds which act as agonists or antagonists to increase or decreasenuclear localisation of an NLS fused to a detectable marker.

[0019] A number of researchers have studied the cell cycle usingtraditional reporter molecules that require the cells to be fixed orlysed. For example Hauser and Bauer (Plant and Soil, 2000, 226, p1-10)used β-glucuronidase (GUS) to study cell division in a plant meristemand Brandeis and Hunt (EMBO J., 1996, vol 15, pp5280-5289) usedchloramphenical acetyl transferase (CAT) fusion proteins to studyvariations in cyclin levels. Although these methods provide a means ofstudying the cell cycle position of a particular cell (using GUS) or theaverage cell cycle status of a population of cells (using CAT) bothmethods are destructive. Neither method allows the repeated analysis ofa specific cell over time and they are therefore not suitable to followthe progression of a cell through the cell cycle.

[0020] None of the preceding methods, which use components of the cellcycle control mechanism, provide means for determining the cell cyclestatus of an individual cell or a population of cells. Consequently,methods are required that enable the cell cycle position of a singleliving cell to be determined non-destructively, allowing the same cellto be repeatedly interrogated over time, and which enable the study ofthe effects of agents having potentially desired or undesired effects onthe cell cycle. Furthermore, it is desirable for cell cycle position tobe determined from a probe controlled directly by cell cycle controlcomponents, rather than indirectly through DNA content or other indirectmarkers of cell cycle position as described above The present inventiondescribes a method which utilises key components of the cell cycleregulatory machinery in defined combinations to provide novel means ofdetermining cell cycle status for individual living mammalian cells in anon-destructive process providing dynamic read out.

[0021] The present invention provides DNA constructs, and cell linescontaining such constructs, that exhibit activation and destruction of adetectable reporter molecule in a cell cycle phase specific manner, bydirect linkage of reporter signal switching to temporal and spatialexpression and destruction of cell cycle components. This greatlyimproves the precision of determination of cell cycle phase status andallows continuous monitoring of cell cycle progression in individualcells. Furthermore, it has been found that key control elements can beisolated and abstracted from functional elements of the cell cyclecontrol mechanism to permit design of cell cycle phase reporters whichare dynamically regulated and operate in concert with, but independentlyof, endogenous cell cycle control components, and hence provide meansfor monitoring cell cycle position without influencing or interferingwith the natural progression of the cell cycle.

SUMMARY OF THE INVENTION

[0022] Accordingly, in a first aspect of the invention, there isprovided a nucleic acid reporter construct comprising a nucleic acidsequence encoding a detectable live-cell reporter molecule operablylinked to and under the control of:

[0023] i) at least one cell cycle phase-specific expression controlelement, and

[0024] ii) a destruction control element;

[0025] wherein said reporter construct is expressed in a mammalian cellat a predetermined position in the cell cycle. Expression being definedas all of the processes involved in the biosynthesis of a protein from agene. It will be further understood that the term ‘live-cell’, as itrelates to reporter molecules, defines a reporter molecule whichproduces a detectable signal in living cells and is therefore suitablefor use in live-cell imaging systems.

[0026] In a second aspect of the Invention, there is provided a methodfor determining the position in the cell cycle of a mammalian cell saidmethod comprising:

[0027] a) expressing in a cell a nucleic acid reporter constructcomprising a nucleic acid sequence encoding a detectable livecell-reporter molecule operably linked to and under the control of:

[0028] i) at least one cell cycle phase-specific expression controlelement, and

[0029] ii) a destruction control element; wherein said reporterconstruct is expressed in a cell at a predetermined point in the cellcycle; and

[0030] b) determining the position in the cell cycle by monitoringsignals emitted by the reporter molecule

[0031] In preferred embodiments of the first and second aspects, thenucleic acid reporter construct is also linked to and under the controlof a cell cycle phase-specific spatial localisation control element

[0032] Suitably, the nucleic acid reporter construct is a DNA construct

[0033] The cell cycle phase-specific expression control element istypically a DNA sequence that controls transcription and/or translationof one or more nucleic acid sequences and permits the cell cyclespecific control of expression. Any expression control element that isspecifically active in one or more phases of the cell cycle may suitablybe used for construction of the cycle position reporter construct.

[0034] Suitably, the cell cycle phase specific expression controlelement may be selected from cell cycle specific promoters and otherelements that influence the control of transcription or translation in acell cycle specific manner. Where the expression control element is apromoter, the choice of promoter will depend on the phase of the cellcycle selected for study.

[0035] Suitable promoters include: cyclin B1 promoter (Cogswell et al,Mol. Cell Biol., (1995), 15(5), 2782-90, Hwang et al, J.Biol.Chem.,(1995), 270(47), 28419-24, Piaggio et al, Exp. Cell Res., (1995),216(2), 396-402); Cdc25B promoter (Korner et al, J.Biol.Chem., (2001),276(13), 9662-9); cyclin A2 promoter (Henglein et al,Proc.Nat.Acad.Sci.USA, (1994), 91(12), 5490-4, Zwicker et al, Embo J.,(1995), 14(18), 4514-22); Cdc2 promoter (Tommasi and Pfeifer, Mol. CellBiol., (1995), 15(12), 6901-13, Zwicker et al, Embo J (1995), 14(18),4514-22), Cdc25C promoter (Korner and Muller, J.Biol.Chem., (2000),275(25), 18676-81, Korner et al, Nucl. Acids Res., (1997), 25(24),4933-9); cyclin E promoter (Botz et al, Mol. Cell Biol., (1996), 16(7),3401-9, Korner and Muller, J.Biol.Chem., (2000), 275(25), 18676-81);Cdc6 promoter (Hateboer et al, Mol. Cell Biol., (1998), 18(11), 6679-97,Yan et al, Proc.Nat.Acad.Sci.USA, (1998), 95(7), 3603-8); DHFR promoter(Shimada et al, J.Biol.Chem., (1986), 261(3), 1445-52, Shimada andNienhuis, J.Biol.Chem., (1985), 260(4), 2468-74) and histones promoters(van Wijnen et al, Proc.Nat.Acad.Sci.USA, (1994), 91, 12882-12886).

[0036] Suitably, the cell cycle phase specific expression controlelement may be selected from cell cycle specific IRES elements and otherelements that influence the control of translation in a cell cyclespecific manner. An IRES element is an internal ribosomal entry sitethat allows the binding of a ribosome and the initiation of translationto occur at a region of mRNA which is not the 5′-capped region. A cellcycle-specific IRES element restricts cap-independent initiation oftranslation to a specific stage of the cell cycle (Sachs, A. B., Cell,(2000), 101, 243-5). Where the expression control element is selected tobe an IRES, suitably its selection will depend on the cell cycle phaseunder study. In this case, a constitutively expressed (eg. CMV or SV40)or inducible (eg. pTet-on pTet-off system, Clontech) promoter may beused to control the transcription of the bicistronic mRNA (Sachs, A. B.,Cell, (2000), 101, 243-5). Alternatively, a non cell cyclephase-dependent IRES element (eg. the EMCV IRES found in pIRES vectors,BD Clontech) may be used in conjunction with a cell cycle specificpromoter element. Alternatively, more precise control of expression ofthe reporter may be obtained by using a cell cycle phase specificpromoter in conjunction with a cell cycle phase specific IRES element.

[0037] IRES elements suitable for use in the invention include: G2-IRES(Cornelis et al, Mol. Cell, (2000), 5(4), 597-605); HCV IRES (Honda etal, Gastroenterology, (2000), 118, 152-162); ODC IRES (Pyronet et al,Mol. Cell, (2000), 5, 607-616); c-myc IRES (Pyronnet et al, Mol. Cell,(2000), 5(4), 607-16) and p58 PITSLRE IRES (Cornelis et al, Mol. Cell,(2000), 5(4), 597-605).

[0038] Table 1 lists some preferred expression control elements that maybe used in accordance with the invention, and indicates the cell cyclephase in which each element is activated. TABLE 1 Cell CyclePhase-Specific Expression Control Elements Element Timing Element TimingCyclin B1 promoter G2 DHFR promoter late G1 Cdc25B promoter S/G2Histones promoters late G1/S Cyclin A2 promoter S G2-IRES G2 Cdc2promoter S HCV IRES M Cdc25C promoter S ODC IRES G2/M Cyclin E promoterlate G1 c-myc IRES M Cdc6 promoter late G1 p58 PITSLRE IRES G2/M

[0039] The destruction control element is a DNA sequence encoding aprotein motif that controls the destruction of proteins containing thatsequence. Suitably, the destruction control element may be cell cyclemediated, for example: Cyclin B1 D-box (Glotzer et al, Nature, (1991),349, 132-138, Yamano et al, EMBO J., (1998), 17(19), 5670-8, Clute andPines, Nature Cell Biology, (1999), 1, 82-87); cyclin A N-terminus (denElzen and Pines, J. Cell Biol., (2001), 153(1), 121-36, Geley et al, J.Cell Biol., (2001), 153, 137-48); KEN box (Pfleger and Kirschner, GenesDev, (2000), 14(6), 655-65), Cyclin E (Yeh et al, Biochem Biophys ResCommun., (2001) 281, 884-90), Cln2 cyclin from S. cerevisiae (Berset etal, Mol. Cell Biol., (2002), pp4463-4476) and p27Kip1 (Montagnoli et al,Genes Dev., (1999), 13(9), 1181-1189, Nakayama et al, EMBO J., (2000),19(9), 2069-81, Tomoda et al, Nature, (1999), 398(6723), 160-5).

[0040] Table 2 lists destruction control elements that may be usedaccording to the invention and indicates the cell cycle phase in whicheach element is activated.

[0041] Alternatively, the destruction control element may be noncell-cycle mediated, such as PEST sequences as described by Rogers etal, Science, (1986), 234, 364-8. Examples of non cell-cycle mediateddestruction control elements include sequences derived from casein,ornithine decarboxylase and proteins that reduce protein half-life. Useof such non cell-cycle mediated destruction control sequences in themethod of the invention provides means for determining the persistencetime of the cell cycle reporter following induction of expression by acell cycle specific promoter. TABLE 2 Destruction Control ElementsElement Timing Cyclin B1 D-box Metaphase through to G1 phase Cyclin AN-terminus Prometaphase through to G1 phase KEN box anaphase/G1 p27Kip1G1 Cyclin E G1/S boundary Cln2 G1/S boundary

[0042] Suitably, the live-cell reporter molecule encoded by the nucleicacid sequence may be selected from the group consisting of fluorescentproteins and enzymes. Preferred fluorescent proteins include GreenFluorescent Protein (GFP) from Aequorea victoria and derivatives of GFPsuch as functional GFP analogues in which the amino acid sequence ofwild type GFP has been altered by amino acid deletion, addition, orsubstitution. Suitable GFP analogues for use in the present inventioninclude EGFP (Cormack, B. P. et al, Gene, (1996), 173, 33-38); EYFP andECFP (U.S. Pat. No. 6,066,476, Tsien, R. et al); F64L-GFP (U.S. Pat. No.6,172,188, Thastrup, O. et al); BFP, (U.S. Pat. No. 6,077,707, Tsien, R.et al). Other fluorescent proteins include DsRed, HcRed and other novelfluorescent proteins (BD Clontech and Labas, Y. A. et al, Proc Natl AcadSci U S A (2002), 99, 4256-61) and Renilla GFP (Stratagene). Suitableenzyme reporters are those which are capable of generating a detectable(e.g. a fluorescent or a luminescent) signal in a substrate for thatenzyme. Particularly suitable enzyme/substrates include:nitroreductase/Cy-Q (as disclosed in WO 01/57237) and β-lactamase/CCF4.

[0043] In a preferred embodiment according to the present invention, thenucleic acid reporter construct may optionally include a cell cyclephase-specific spatial localisation control element comprising a DNAsequence encoding a protein motif that is capable of controlling thesub-cellular localisation of the protein in a cell cycle specificmanner. Such a localisation control element may be used advantageouslyaccording to the invention where:

[0044] i) a specific sub-cellular localisation of the reporter isdesirable; and/or

[0045] ii) more precise determination of the cell cycle position isrequired.

[0046] It may be required to determine the sub-cellular localisation ofthe reporter either to ensure its effective operation and/ordestruction. More precise determination of the cell cycle position maybe possible using a localisation control element since this will permitmeasurement of both intensity and location of the reporter signal.

[0047] Suitable spatial localisation control elements include those thatregulate localisation of a cell cycle control protein, for example thecyclin B1 CRS.

[0048] The term, operably linked indicates that the elements arearranged so that they function in concert for their intended purposes,e.g. transcription initiates in a promoter and proceeds through the DNAsequence coding for the fluorescent protein of the invention. FIG. 2(2A/2B/2C) illustrates the general construction of a DNA constructaccording to the invention.

[0049] In a preferred aspect of the invention, the construct comprises acyclin B1 promoter, a cyclin B1 destruction box (D-box), a cyclin B1cytoplasmic retention sequence (CRS) and a green fluorescent protein(GFP).

[0050] In a particular example according to the present invention, thenucleic acid reporter construct comprises an expression vectorcomprising the following elements:

[0051] a) a vector backbone comprising:

[0052] i) a bacterial origin of replication; and

[0053] ii) a bacterial drug resistance gene;

[0054] b) a cell cycle phase specific expression control element;

[0055] c) a destruction control element; and

[0056] d) a nucleic acid sequence encoding a reporter molecule.

[0057] Optionally, the nucleic acid reporter construct additionallycontains a cell cycle phase-specific spatial localisation controlelement and/or a eukaryotic drug resistance gene, preferably a mammaliandrug resistance gene.

[0058] Expression vectors may also contain other nucleic acid sequences,such as polyadenylation signals, splice donor/splice acceptor signals,intervening sequences, transcriptional enhancer sequences, translationalenhancer sequences and the like. Optionally, the drug resistance geneand the reporter gene may be operably linked by an internal ribosomeentry site (IRES), which is either cell cycle specific (Sachs, et al,Cell, (2000), 101, 243-245) or cell cycle independent (Jang et al, J.Virology, (1988), 62, 2636-2643 and Pelletier and Sonenberg, Nature,(1988), 334, 320-325), rather than the two genes being driven fromseparate promoters. When using a non cell-cycle specific IRES elementthe pIRES-neo and pIRES-puro vectors commercially available fromClontech may be used.

[0059] In a particular embodiment of the first aspect, the nucleic acidreporter construct is assembled from a DNA sequence encoding the cyclinB1 promoter operably linked to DNA sequences encoding 171 amino acids ofthe amino terminus of cyclin B1 and a DNA sequence encoding a greenfluorescent protein (GFP) (FIG. 3). Motifs controlling the localisationand destruction of cyclin B1 have all been mapped to ^(˜)150 amino acidsin the amino terminus of the molecule. Consequently, an artificial cellcycle marker can be constructed using only sequences from the aminoterminus of cyclin B1, which will not interfere with cell cycleprogression since it lacks a specific sequence, termed the cyclin box,(Nugent et al, J. Cell. Sci., (1991), 99, 669-674) which is required tobind to and activate a partner kinase. Key regulatory motifs requiredfrom the amino terminus sequence of cyclin B1 are: i) a nine amino acidmotif termed the destruction box (D-box). This is necessary to targetcyclin B1 to the ubiquitination machinery and, in conjunction with atleast one C-terminal lysine residue, this is also required for itscell-cycle specific degradation; ii) an approximately ten amino acidnuclear export signal (NES). This motif is recognised, either directlyor indirectly, by exportin 1 and is sufficient to maintain the bulk ofcyclin B1 in the cytoplasm throughout interphase; iii) approximatelyfour mitosis-specific phosphorylation sites that are located in andadjacent to the NES and confer rapid nuclear import and a reducednuclear export at mitosis. When expressed in a eukaryotic cell, theconstruct will exhibit cell cycle specific expression and destruction ofthe GFP reporter which parallels the expression and degradation ofendogenous cyclin B1. Hence, measurement of GFP fluorescence intensitypermits identification of cells in the G2/M phase of the cell cycle(FIG. 4). Furthermore, since the fluorescent product of the constructwill mimic the spatial localisation of endogenous cyclin B1, analysis ofthe sub-cellular distribution of fluorescence permits further precisionin assigning cell cycle position. At prophase, cyclin B1 rapidlytranslocates into the nucleus, consequently the precise localisation ofGFP fluorescence in the cell can be used to discriminate cellstransitioning from interphase to mitosis. Once a cell reaches metaphase,and the spindle assembly checkpoint is satisfied, cyclin B1 is veryrapidly degraded, and consequently the disappearance of GFP fluorescencecan be used to identify cells at mid-M phase.

[0060] Expression of the construct in a population of unsynchronisedcells will result in each cell exhibiting cyclical expression anddestruction of the fluorescent product from the construct, resulting ina continuous blinking pattern of fluorescence from all cells in thepopulation. Analysis of the fluorescence intensity of each cell withtime consequently yields dynamic information on the cell cycle status ofeach cell as illustrated in FIG. 4.

[0061] Further embodiments of the nucleic acid reporter constructaccording to the first aspect may be constructed by selecting suitablealternative cell cycle control elements, for example from those shown inTables 1 and 2, to design cell cycle phase reporters which report adesired section of the cell cycle.

[0062] The construction and use of expression vectors and plasmids arewell known to those of skill in the art. Virtually any mammalian cellexpression vector may be used in connection with the cell cycle markersdisclosed herein. Examples of suitable vector backbones which includebacterial and mammalian drug resistance genes and a bacterial origin ofreplication include, but are not limited to: pCI-neo (Promega), pcDNA(Invitrogen) and pTriEx1 (Novagen). Suitable bacterial drug resistancegenes include genes encoding for proteins that confer resistance toantibiotics including, but not restricted to: ampicillin, kanamycin,tetracyclin and chloramphenicol. Eurkaryotic drug selection markersinclude agents such as: neomycin, hygromycin, puromycin, zeocin,mycophenolic acid, histidinol, gentamycin and methotrexate.

[0063] The DNA construct may be prepared by the standard recombinantmolecular biology techniques of restriction digestion, ligation,transformation and plasmid purification by methods familiar to thoseskilled in the art and are as described in Sambrook, J. et al (1989),Molecular Cloning—A Laboratory Manual, Cold Spring Harbor LaboratoryPress. Alternatively, the construct can be prepared synthetically byestablished methods, eg. the phosphoramidite method described byBeaucage and Caruthers, (Tetrahedron Letters, (1981), 22, 1859-1869) orthe method described by Matthes et al (EMBO J., (1984), 3, 801-805).According to the phosphoramidite method, oligonucleotides aresynthesised, eg. in an automatic DNA synthesizer, purified, annealed,ligated and cloned into suitable vectors. The DNA construct may also beprepared by polymerase chain reaction (PCR) using specific primers, forinstance, as described in U.S. Pat. No. 4,683,202 or by Saiki et al(Science, (1988), 239, 487-491). A review of PCR methods may be found inPCR protocols, (1990), Academic Press, San Diego, Calif., U.S.A.

[0064] During the preparation of the DNA construct, the gene sequenceencoding the reporter must be joined in frame with the cell cycle phasespecific destruction control element and optionally the spatiallocalisation control element. The resultant DNA construct should then beplaced under the control of one or more suitable cell cycle phasespecific expression control elements.

[0065] In a third aspect, there is provided a host cell transfected witha nucleic acid reporter construct according to the present invention.The host cell into which the construct or the expression vectorcontaining such a construct is introduced, may be any mammalian cellwhich is capable of expressing the construct.

[0066] The prepared DNA reporter construct may be transfected into ahost cell using techniques well known to the skilled person. Oneapproach is to temporarily permeabilise the cells using either chemicalor physical procedures. These techniques may include: electroporation(Tur-Kaspa et al, Mol. Cell Biol. (1986), 6, 716-718; Potter et al,Proc.Nat.Acad.Sci.USA, (1984), 81, 7161-7165), a calcium phosphate basedmethod (eg. Graham and Van der Eb, Virology, (1973), 52, 456-467 andRippe et al, Mol. Cell Biol., (1990), 10, 689-695) or directmicroinjection.

[0067] Alternatively, cationic lipid based methods (eg. the use ofSuperfect (Qiagen) or Fugene6 (Roche) may be used to introduce DNA intocells (Stewart et al, Human Gene Therapy, (1992), 3, 267; Torchilin etal, FASEB J, (1992), 6, 2716; Zhu et al, Science, (1993), 261, 209-211;Ledley et al, J. Pediatrics, (1987), 110, 1; Nicolau et al, Proc.Nat.Acad.Sci.,USA, (1983), 80, 1068; Nicolau and Sene, Biochem.Biophys.Acta,(1982), 721, 185-190). Jiao et al, Biotechnology, (1993), 11, 497-502)describe the use of bombardment mediated gene transfer protocols fortransferring and expressing genes in brain tissues which may also beused to transfer the DNA into host cells.

[0068] A further alternative method for transfecting the DNA constructinto cells, utilises the natural ability of viruses to enter cells. Suchmethods include vectors and transfection protocols based on, forexample, Herpes simplex virus (U.S. Pat. No. 5,288,641), cytomegalovirus(Miller, Curr. Top. Microbiol. Immunol., (1992), 158, 1), vaccinia virus(Baichwal and Sugden, 1986, in Gene Transfer, ed. R. Kucherlapati, NewYork, Plenum Press, p117-148), and adenovirus and adeno-associated virus(Muzyczka, Curr. Top. Microbiol. Immunol., (1992), 158, 97-129).

[0069] Examples of suitable recombinant host cells include HeLa cells,Vero cells, Chinese Hamster ovary (CHO), U2OS, COS, BHK, HepG2, NIH 3T3MDCK, RIN, HEK293 and other mammalian cell lines that are grown invitro. Such cell lines are available from the American Tissue CultureCollection (ATCC), Bethesda, Md., U.S.A. Cells from primary cell linesthat have been established after removing cells from a mammal followedby culturing the cells for a limited period of time are also intended tobe included in the present invention.

[0070] Cell lines which exhibit stable expression of a cell cycleposition reporter may also be used in establishing xenografts ofengineered cells in host animals using standard methods. (Krasagakis, K.J et al, Cell Physiol., (2001), 187(3), 386-91; Paris, S. et al,Clin.Exp.Metastasis, (1999), 17(10), 817-22). Xenografts of tumour celllines engineered to express cell cycle position reporters will enableestablishment of model systems to study tumour cell division, stasis andmetastasis and to screen new anticancer drugs.

[0071] Use of engineered cell lines or transgenic tissues expressing acell cycle position reporter as allografts in a host animal will permitstudy of mechanisms affecting tolerance or rejection of tissuetransplants (Pye D and Watt, D. J., J. Anat., (2001), 198 (Pt 2),163-73; Brod, S. A. et al, Transplantation (2000), 69(10), 2162-6).

[0072] To perform the method for determining the cell cycle position ofa cell according to the second aspect, cells transfected with the DNAreporter construct may be cultured under conditions and for a period oftime sufficient to allow expression of the reporter molecule at aspecific stage of the cell cycle. Typically, expression of the reportermolecule will occur between 16 and 72 hours post transfection, but mayvary depending on the culture conditions. If the reporter molecule isbased on a green fluorescent protein sequence the reporter may take adefined time to fold into a conformation that is fluorescent. This timeis dependent upon the primary sequence of the green fluorescent proteinderivative being used. The fluorescent reporter protein may also changecolour with time (see for example, Terskikh, Science, (2000), 290,1585-8) in which case imaging is required at specified time intervalsfollowing transfection.

[0073] The cell cycle position of the cells may be determined bymonitoring the expression of the reporter molecule and detecting signalsemitted by the reporter using an appropriate detection device. If thereporter molecule produces a fluorescent signal, then, either aconventional fluorescence microscope, or a confocal based fluorescencemicroscope may be used. If the reporter molecule produces luminescentlight, then a suitable device such as a luminometer may be used. Usingthese techniques, the proportion of cells expressing the reportermolecule may be determined. If the DNA construct contains translocationcontrol elements and the cells are examined using a microscope, thelocation of the reporter may also be determined. In the method accordingto the present invention, the fluorescence of cells transformed ortransfected with the DNA construct may suitably be measured by opticalmeans in for example; a spectrophotometer, a fluorimeter, a fluorescencemicroscope, a cooled charge-coupled device (CCD) imager (such as ascanning imager or an area imager), a fluorescence activated cellsorter, a confocal microscope or a scanning confocal device, where thespectral properties of the cells in culture may be determined as scansof light excitation and emission.

[0074] In the embodiment of the invention wherein the nucleic acidreporter construct comprises a drug resistance gene, followingtransfection and expression of the drug resistance gene (usually 1-2days), cells expressing the modified reporter gene may be selected bygrowing the cells in the presence of an antibiotic for which transfectedcells are resistant due, to the presence of a selectable marker gene.The purpose of adding the antibiotic is to select for cells that expressthe reporter gene and that have, in some cases, integrated the reportergene, with its associated promoter, IRES elements, enhancer andtermination sequences into the genome of the cell line. Followingselection, a clonal cell line expressing the construct can be isolatedusing standard techniques. The clonal cell line may then be grown understandard conditions and will express reporter molecule and produce adetectable signal at a specific point in the cell cycle.

[0075] Cells transfected with the nucleic acid reporter constructaccording to the present invention may be grown in the absence and/orthe presence of a test system to be studied and whose effect on the cellcycle of a cell is to be determined. By determining the proportion ofcells expressing the reporter molecule and, optionally, the localisationof the signal within the cell, it is possible to determine the effect ofthe test system on the cell cycle of the cells, for example, whether thetest system arrests the cells in a particular stage of the cell cycle,or whether the effect is to speed up or slow down cell division.

[0076] Thus, in a fourth aspect, there is provided a method ofdetermining the effect of a test system on the cell cycle position of amammalian cell, said method comprising:

[0077] a) expressing in a cell in the absence and In the presence ofsaid test system a nucleic add reporter construct comprising a nucleicacid sequence encoding a detectable live-cell reporter molecule operablylinked to and under the control of:

[0078] i) at least one cell cycle phase-specific expression controlelement, and

[0079] ii) a destruction control element; wherein said reporterconstruct is expressed in a cell at a predetermined point in the cellcycle; and

[0080] b) determining the cell cycle position by monitoring signalsemitted by the reporter molecule wherein a difference between theemitted signals measured In the absence and in the presence of said testsystem is indicative of the effect of said test system on the cell cycleposition of said cell.

[0081] In a fifth aspect, there is provided a method of determining theeffect of a test system on the cell cycle position of a mammalian cell,the method comprising:

[0082] a) expressing in the cell in the presence of the test system anucleic acid reporter construct comprising a nucleic acid sequenceencoding a detectable live-cell reporter molecule operably linked to andunder the control of:

[0083] i) at least one cell cycle phase-specific expression controlelement, and

[0084] ii) a destruction control element;

[0085] wherein the reporter construct is expressed in a cell at apredetermined point in the cell cycle; and

[0086] b) determining the cell cycle position by monitoring signalsemitted by the reporter molecule,

[0087] c) comparing the emitted signal in the presence of the testsystem with a known value for the emitted signal in the absence of thetest system;

[0088] wherein a difference between the emitted signal measured in thepresence of the test system and the known value in the absence of thetest system is indicative of the effect of the test system on the cellcycle position of the cell.

[0089] In a sixth aspect, there is provided a method of determining theeffect of a test system on the cell cycle position of a mammalian cell,said method comprising:

[0090] a) providing cells containing a nucleic acid reporter constructcomprising a nucleic acid sequence encoding a detectable live-cellreporter molecule operably linked to and under the control of

[0091] i) at least one cell cycle phase-specific expression controlelement, and

[0092] ii) a destruction control element;

[0093] wherein the reporter construct is expressed in a cell at apredetermined point in the cell cycle;

[0094] b) culturing first and second populations of the cellsrespectively in the presence and absence of a test system and underconditions permitting expression of the nucleic acid reporter construct;and

[0095] c) measuring the signals emitted by the reporter molecule in thefirst and second cell populations;

[0096] wherein a difference between the emitted signals measured in thefirst and second cell populations is indicative of the effect of thetest system on the cell cycle position of the cell.

[0097] By the term test system, it is intended to mean an agent such asa drug, hormone, protein, peptide, nucleic acid and the like, to whichthe cell is exposed. Alternatively, the test system may be an agent suchas a nucleic acid, peptide or protein that is expressed in the cellunder study. For example, cells transfected with the nucleic acidreporter constructs according to the present invention may be used todetermine whether expression of cDNA containing constructs encodingproteins under study have an effect on the cell cycle position of acell. A series of cDNAs, inserted into a mammalian expression vector,may be transiently transfected into a cell stably expressing the cellcycle position reporter. By monitoring the expression and location ofthe nucleic acid reporter construct in these transfected cells, it ispossible to determine the effects of the proteins encoded by the cDNAson the cell cycle.

[0098] The cell cycle position nucleic acid reporter constructsaccording to the present invention may also be used in a method todetermine the effect of the cell cycle position on a cellular process,or to determine the effect of the cell cycle position on the action of atest substance on a cellular process. It is well known that manycellular processes, including those that respond to external stimuli,are influenced by the cell cycle so as to operate or respond differentlyat different stages of the cell cycle. For example, endothelin receptorshave been shown to be expressed at different levels during differentphases of the cell cycle (Okazawa etal. J.Biol.Chem., (1998), 273,12584-12592) and consequently the sensitivity of cells to endothelininduced apoptosis varies with the cell cycle position. Similarly,cellular Ca²⁺ mobilisation responses to vasopressin differ according tocell cycle position (Abel et al. J.Biol.Chem., (2000), 275, 32543-32551)due to variations in the route of signal transduction which utilisedifferent G-proteins at different cell cycle phases. Use of the cellcycle position reporter constructs will allow cell to cell variations ina biological assay, measured using an appropriate assay reporter, to becorrelated with the signal from a cell cycle position reporter in orderto determine if any variations in the assay signal correlate with thecell cycle position reporter signal and hence determine any cell cycledependence of the assay signal. For example, assays may be devised inwhich the amount of a red fluorescently labelled ligand bound to a cellsurface receptor is correlated with cell cycle status determined using aGFP cell cycle position reporter. By cellular process, it is meant thenormal processes which living cells undergo and includes: biosynthesis,uptake, transport, receptor binding, metabolism, fusion, biochemicalresponse, growth and death.

[0099] Two or more cell cycle position nucleic acid reporter constructsmay be used in combination in applications that include reporting ontransition through two or more, cell cycle phases within the same cell.To achieve such duplex or multiplex reporting, two or more differentconstructs are engineered and expressed in the same cell, wherein eachreporter construct comprises a different combination of control elementslinked to a different and distinguishable reporter. For example,cellular expression of a construct comprising a cyclin B1 promoter andcyclin B1 D-box operably linked to GFP in combination with expression inthe same cell of a second construct comprising a cyclin A2 promoter andcyclin B1 D-box operably linked to blue fluorescent protein (BFP) willallow discrimination of cells in S phase (blue fluorescence) from cellsin G2/M phase (blue and green fluorescence).

[0100] The cell cycle position nucleic acid reporter constructs andassay methods according to the present invention may be used in avariety of additional applications, for example:

[0101] i) Cells transfected with the cell cycle position reporterconstructs of the present invention may be used to determine the effectof the cell cycle on the expression, translocation or subcellulardistribution of a second marker in a multiplexed assay. For example, ifthe Intracellular translocation of a fluorescent reporter to the plasmamembrane is being studied and it is found that a test compound resultsin translocation in a percentage of the cells, then, using cellstransfected with a construct according to the invention, it is possibleto determine whether the translocation was cell cycle dependent.

[0102] Thus, in a seventh aspect of the invention there is provided amethod of determining the effect of the mammalian cell cycle on theexpression, translocation or sub-cellular distribution of a firstdetectable reporter which is known to vary in response to a test system,the method comprising:

[0103] a) expressing in the cell in the presence of the test system asecond nucleic acid reporter construct comprising a nucleic acidsequence encoding a detectable live-cell reporter molecule operablylinked to and under the control of:

[0104] i) a cell cycle phase-specific expression control element, and

[0105] ii) a destruction control element; wherein the reporter constructis expressed in a cell at a predetermined point in the cell cycle;

[0106] b) determining the cell cycle position by monitoring signalsemitted by the second reporter molecule;

[0107] c) monitoring the signals emitted by the first detectablereporter,

[0108] wherein the relationship between cell cycle position determinedby step b) and the signal emitted by the first detectable reporter isindicative of whether or not the expression, translocation orsub-cellular distribution of the first detectable reporter is cell cycledependent. The term ‘test system’ is to be understood as hereinbeforedescribed in relation to the fourth, fifth and sixth aspect of thepresent invention.

[0109] ii) Cell cycle position reporters of the present invention may beused in combination with analysis of endogenous cellular markers orphenomena that are cell cycle related, in order to gain furtherinformation on the cell cycle status of an individual cell or a cellpopulation. For example, it is well known that the Golgi complex has adistinctive morphology in mammalian cells, comprising a ribbon likestructure adjacent to the nucleus, and that distinctive changes occur inthe structure of the Golgi as cells undergo mitosis as the ribbonstructure is converted to clusters of vesicles and tubules dispersedthroughout the mitotic cell. (Lowe et al., Trends Cell Biol., (1998),8(1) 40-44). Analysis of the morphology of cell components, such as theGolgi apparatus, which vary in a known fashion with cell cycleprogression, for example by use of specific fluorescent stains, may beused in combination with a cell cycle position reporter to enable moredetailed analysis of cell cycle progression.

[0110] iii) The cell cycle position reporters according to the presentinvention may also be used for monitoring cell cycle status andprogression in in-vivo applications. For example, the introduction of aDNA construct encoding a cell cycle reporter into living specimens suchas Xenopus oocytes, and living organisms such as C. elegans andDrosophila, via transfection of individual cells or multiple cells canbe achieved by microinjection (Krone P. H., and Heikkila J. J.,Development, (1989), 106(2), 271-81), ballistic injection (Horard B. etal., Insect Mol.Biol., (1994), 3(4), 61-5), and other methods well knownto the skilled person. Introduction of cell cycle reporter constructsinto such specimens will enable investigation of cell cycle progressionand control in cell progeny of transfected cells. Information fromreporters is likely to be of significant value in study of growth anddevelopment in model organisms.

[0111] iv) The reporters of the present invention may be used in thegeneration of transgenic organisms, ie. where the DNA encoding the cellcycle position reporter is stably expressed in all cells of an organismor animal. Such transgenic organisms may be generated by microinjectingDNA into an early embryo, generally into one of the pronuclei of a newlyfertilized egg. (Bishop J. O., Reprod.Nutr.Dev., (1996), 36(6), 607-18).Transgenic techniques may be used to engineer cell cycle positionreporters into a range of host species from simple organisms such as C.elegans (Daniells, C. et al, Mutat.Res., (1998), 399(1), 55-64) to morecomplex organisms such as mice and rats (Sills, R. C., et al.,Toxicol.Lett., (2001), 20(1-3), 187-98). Establishment of stabletransgenic expression of a cell cycle position reporter in all cells ofa transgenic organism will allow cell cycle status to be determined inany cell type within, or isolated from, the organism, including culturedcell lines derived from the organism. Accordingly, in an eighth aspectof the present invention there is provide a transgenic organismcomprising a cell as hereinbefore described.

[0112] v) Cell lines that exhibit stable expression of a cell cycleposition reporter may also be used in establishing xenografts ofengineered cells in host animals using standard methods. (Krasagakis, K.J et al, Cell Physiol., (2001), 187(3), 386-91; Paris, S. et al, Clin.Exp. Metastasis, (1999), 17(10), 817-22). Thus, in a ninth aspect of thepresent invention, there is provided a cell line comprising a cell ashereinbefore described for use in establishing xenografts in a hostorganism. Xenografts of tumour cell lines engineered to express cellcycle position markers will enable establishment of model systems tostudy tumour cell division, stasis and metastasis and to screen newanticancer drugs.

[0113] vi) Use of engineered cell lines or transgenic tissues expressinga cell cycle position reporter as allografts in a host animal willpermit study of mechanisms affecting tolerance or rejection of tissuetransplants (Pye, D. and Watt, D. J., J. Anat., (2001), 198(Pt 2),163-73; Brod, S. A. et al, Transplantation (2000), 69(10), 2162-6).Therefore, in a tenth aspect of the present invention there is provideda cell line comprising a cell as hereinbefore described for use inestablishing allografts in a host organism.

[0114] In an eleventh aspect of the present invention there is provideda transgenic organism comprising a cell as hereinbefore described.

BRIEF DESCRIPTION OF THE INVENTION

[0115] The invention is further illustrated by reference to thefollowing examples and figures in which:

[0116]FIG. 1 is a schematic diagram illustrating cyclin B1 regulationduring cell cycle progression. The cell cycle proceeds in the directionof the arrow with cyclin B1 expression driven by a cell cyclephase-specific promoter which initiates expression at the end of the Sphase and peaks during G2 (A). At the start of mitosis (B) cyclin B1translocates from the cytoplasm to the nucleus and from metaphaseonwards (C) the protein is specifically degraded.

[0117]FIG. 2 is a schematic diagram illustrating cell cycle positionnucleic acid reporter constructs according to the present invention andin which, 2A utilises a cell cycle phase-specific promoter and no IRESelement, 2B utilises an IRES element to facilitate mammalian selection,and 2C contains a constituitive or inducible mammalian promoter and acell cycle phase-specific IRES as the expression control element. Ineach case A represents a cell cycle phase-specific expression control(promoter), B represents a cell cycle phase specific destruction controlelement, C represents a cell cycle phase specific localisation controlelement, D represents a reporter gene, E represents a non-cell cyclespecific IRES element, F represents a mammalian selectable marker, Grepresents a mammalian constitutive promoter and H represents a cellcycle specific IRES element.

[0118]FIG. 3 shows a DNA construct for determining the G2/M phase of thecell cycle, the construct containing a cyclin B1 promoter (A), cyclin B1destruction box (D-box) (B), cyclin B1 CRS (C) and a GFP reporter (D).

[0119]FIG. 4 illustrates the expression of a nucleic acid constructexpressing the G2/M cell cycle phase marker in a population ofunsynchronised cells. Each cell exhibits cyclical expression anddestruction of the fluorescent product from the construct, resulting ina continuous “blinking” pattern of fluorescence from all cells in thepopulation. Analysis of the fluorescence intensity of each cell at times1, 2, 3 and 4 yields dynamic information on the cell position status ofeach cell.

[0120]FIG. 5 is a bar chart showing the effect of cell cycle blockingagents on GFP fluorescence from a cell cycle phase marker according tothe invention. A represents cells inhibited in mitosis bydemecolchicine, B represents control cells and C represents cellsinhibited at G1/S phase by mimosine;

[0121]FIG. 6 is a series of time lapse photographs showing a cell thathas been microinjected with the construct described in Example 1 andundergoing mitosis according to Example 3.

[0122]FIG. 7 is a series of time lapse photographs showing a U2-OS cellexpressing the construct described in Example 1 undergoing mitosisaccording to Example 4. In panel A the cell on the left is in G2-phaseof the cell cycle, in panel B the cell has entered prophase, in panel Cthe cell is in metaphase, in panel D the cell is in telophase and inpanel E the two daughter cells are non-fluorescent and in G1 phase.

[0123]FIG. 8 is a graph showing the relative fluorescence of a U2-OScell and its progeny that are stably expressing the construct describedin Example 1 according to Example 4. The cell undergoes mitosis (A)after 4 hours and divides into two daughter cells (1,2). Daughter 1 thenundergoes mitosis (B) at 34 hours dividing into two granddaughters (1.1and 1.2) and Daughter 2 undergoes mitosis at 42 hours (C) dividing intogranddaughters (2.1 and 2.2). The bold arrows show the increase influorescence of the daughter cells during G2-phase and prior to mitosis.

[0124]FIG. 9 is a FACS analysis of a U2-OS cell line that stablyexpresses an eGFP containing construct according to Example 5. The topgraph shows a histogram of propidium iodide staining with FL3A (redfluorescence) plotted on the X-axis against number of cells with thatfluorescence on the Y-axis. This graph illustrates that the proportionof cells in G1, S and G2/M are as expected. The bottom graph is adot-plot of the same cells showing FL3A (red) on the X-axis and FL1 H(green fluorescence) on the Y-axis. The diagonal pattern (boxed)indicates that cells in G2/M have more green fluorescence than S phasecells which in turn has more fluorescence than G1 phase cells.

[0125]FIG. 10 is a FACS analysis showing the effect of cell cycleinhibitors upon the relative green fluorescence intensity of the stablecell line described in Example 5 according to Example 6. As in FIG. 9the histograms (on the left) show number of cells (Y-axis) against FL3Aand the dot-plots (on the right) show the same cells plotted with FL1 H(Y-axis) against FL3A (X-axis). The top two graphs show control cellsthat have not been treated with a cell cycle inhibitor. As can be seenthese cells show the typical cell cycle profile (A) and have a diagonalpattern indicating that cells with more GFP are in the G2/M part of thecell cycle. The middle two graphs show cells that have been blocked inG2/M by colchicine (C). The majority of these cells have a relativelyhigh green fluorescence (D). The bottom two graphs show cells that havebeen partially blocked in G1/S by mimosine (E). The majority of thesecells have a relatively low green fluorescence (F).

DETAILED DESCRIPTION OF THE INVENTION EXAMPLES

[0126] 1. Preparation of DNA Construct

[0127] i) The N-terminal third of the cyclin B1 mRNA (amino acids1-171), encoding the cyclin B1 destruction box and the NES was amplifiedwith HindIII and BamHI ends using standard PCR techniques and thefollowing primers:GGGAAGCTTAGGATGGCGCTCCGAGTCACCAGGAACGCCGGATCCCACATATTCACTACAAAGGTT.

[0128] ii) The gene for wtGFP was amplified with primers designed tointroduce restriction sites that would facilitate construction of fusionproteins. The PCR product was cloned into pTARGET (Promega) according tomanufacturer's instructions and mutations (F64L/S175G/E222G) wereintroduced using the QuikChange site-directed mutagenesis kit(Stratagene). Constructs were verified by automated DNA sequencing. DNAencoding the mutant GFP was then cloned downstream of the cyclin B1N-terminal region using BamHI and SaII restriction sites.

[0129] iii) The cell cycle dependent region of the cyclin B1 promoter(−150->+182) was amplified with SacII and HindIII sites and clonedupstream of the Cyclin B1 N-terminal region and the GFP fusion protein.

[0130] iv) The promoter and recombinant protein encoding DNA was excisedand cloned in place of the CMV promoter in a BgIII/NheI cut pCI-Neoderived vector.

[0131] 2. Effect of Cell Cycle Blocking Agents on GFP Fluorescence fromCell Cycle Phase Marker Using Transiently Transfected Cells.

[0132] U2OS cells (ATCC HTB-96) were cultured in wells of a 96 wellmicrotitre plate. Cells were transfected with a cell cycle reporterconstruct prepared according to Example 1, comprising a cyclin B1promoter operably linked to sequences encoding the cyclin B1 D-box, thecyclin B1 CRS, and GFP in a pCORON4004 vector (Amersham Biosciences)using Fugene 6 (Roche) as the transfection agent.

[0133] Following 24 hours of culture, cells were exposed to the specificcell cycle blockers mimosine (blocks at G1/S phase boundary) ordemecolcine (blocks in M phase). Control cells were exposed to culturemedia alone.

[0134] Cells were incubated for a further 24 hours and then analysed fornuclear GFP expression using a confocal scanning imager with automatedimage analysis (IN Cell Analysis System, Amersham Biosciences).

[0135] As shown in FIG. 5, cells exposed to demecolcine showed increasedfluorescence compared to control cells while cells exposed to mimosineshowed decreased fluorescence compared to control cells. These resultsare consistent with the proposed use of the cell cycle phase reporter ofthe invention. Cells blocked in G1/S phase (mimosine treated), prior tothe time of activation of the cyclin B1 promoter, show reducedfluorescence, while cells blocked in M phase (demecolcine treated),prior to the time of action of the cyclin B1 D-box, show increasedfluorescence.

[0136] These results indicate that cell cycle phase reporters of thecurrent invention are suitable for detecting agents which modulate cellcycle progression in a transient system and furthermore such reporterspermit identification of the phase of the cell cycle in which cells areblocked.

[0137] 3. Microinjection and Time-Lapse Photography of the Construct

[0138] HeLa cells were micro-injected with the construct preparedaccording to Example 1 and examined by time lapse microscopy, as shownin FIG. 6. Differential interference contrast (DIC) images are shown onthe left with the corresponding fluorescence image on the right. Frame Ashows a cell (arrowed) in metaphase which shows bright fluorescence inthe nucleus. Frames B and C show the same cell at later times inanaphase (B) and late anaphase (C). The DIC images of B and C show thedivision of the cell into two daughter cells (indicated by 2 arrows),the corresponding fluorescence images show the loss of fluorescenceaccompanying destruction of the fluorescent construct as the cell cycleprogresses.

[0139] 4. Stable Cell Line Production and Time Lapse Photography

[0140] U2-OS cells (ATCC HTB-96) were transfected with the constructdescribed in example 1 and grown for several months in culture mediacontaining 1 mg/ml geneticin to select for cells stably expressing theconstruct. A number of clones were picked by standard methods (e.g.described in Freshney, Chapter 11 in Culture of Animal Cells, (1994)Wiley-Liss Inc) and a clone containing fluorescent cells was isolated.This cell line was maintained at 37° C. in culture media containing 25mM HEPES and a fluorescence and transmitted image of the cells takenevery 15 minutes over a period of 24 hours using a standard xenon lampat 488 nm. FIG. 7 shows 5 frames from a portion of the image thatindicates that the cell line is behaving as expected. Cells in G2exhibit green fluorescence in the cytoplasm, cells in early mitosis havefluorescence predominantly in the nucleus and following mitosis thereporter gene is degraded and the cells lose their fluorescence.

[0141]FIG. 8 shows the fate of a cell from the same clone that wasmonitored over 48 hours and that underwent two cell divisions to producefour granddaughter cells. For each time point the average intensity ofeach of the cells' progeny is measured and plotted against time. As canbe seen the original cell enters mitosis at ^(˜)4 hours, one of thedaughters divides at 32 hours and the other at 42 hours into theexperiment. As cells leave S-phase and enter G2 there is a steadyincrease in average intensity until the cell enters mitosis when thecell rounds up and the average intensity increases dramatically.

[0142] 5. Preparation of a Brighter Stable Cell Line and Subsequent FACSAnalysis

[0143] The green fluorescent protein reporter sequence in the vectordescribed in example 1 was replaced with enhanced GFP (EGFP; Cormack, B.P. et al, Gene, (1996), 173, 33-38; BD Clontech) by standard methods.The EGFP gene is a brighter form of GFP containing the mutations F64Land S65T. In addition, EGFP contains codons that have been altered tooptimise expression in mammalian cells. This new construct wastransfected into U2-OS cells and a number of colonies were isolated byselection with geneticin followed by sorting of single cells using afluorescence activated cell sorter. These clones showed brighterfluorescence than those generated in example 4 and as expectedfluorescence intensity and location appeared to vary according to thecell cycle phase of the cell.

[0144] The cells were prepared for FACS analysis by standard methods.Briefly the cells were fixed and permeabilised using CytoFix/CytoPerm(Becton Dickinson) according to the manufacturers procedures. The cellswere then treated with 50 μg/ml RNAse and 0.4% Triton X-100 andcounterstained with 100 μg/ml propidium iodide. The degree of propidiumiodide staining is proportional to the amount of DNA in the cell andtherefore a measure of the cell cycle phase of the cell. As can be seenin FIG. 9, as expected, the degree of red propidium iodide staining andthe amount of green GFP fluorescence appear to be proportional in thecells.

[0145] 6. The Effect of Cell Cycle Inhibiting Drugs On GFP ExpressionLevels

[0146] The cells prepared in Example 5 were grown in 25 cm² flasks andtreated with either 100 ng/ml demecolcine (Sigma) or 1 mM mimosine(Sigma) for 24 hours. The cells were then fixed, permeabilised andstained with propidium iodide as described in example 5. FACS analysisrevealed that, as expected, cells treated with the colchicine analoguearrested in G2/M and cells treated with mimosine arrested at the G1/Sboundary. As is also expected the cells that had been arrested in G2/Mwere brighter than the cells that had been arrested at G1/S (FIG. 10).

1 2 1 36 DNA artificial sequence synthetic oligonucleotide 1 gggaagcttaggatggcgct ccgagtcacc aggaac 36 2 30 DNA artificial sequence syntheticoligonucleotide 2 gccggatccc acatattcac tacaaaggtt 30

1: A nucleic acid reporter construct comprising a nucleic acid sequenceencoding a detectable live-cell reporter molecule operably linked to andunder the control of: i) at least one cell cycle phase-specificexpression control element, and ii) a destruction control element;wherein said reporter construct is expressed in a mammalian cell at apredetermined position in the cell cycle. 2: The construct of claim 1,wherein said expression control element controls transcription in a cellcycle specific manner. 3: The construct of claim 1, wherein theexpression control element controls translation in a cell cycle specificmanner. 4: The construct of claim 1, wherein the expression controlelement is selected from a cell cycle specific promoter and a cell cyclespecific IRES. 5: The construct of claim 4, wherein said promoter isselected from the group consisting of cyclin B1 promoter, Cdc25Bpromoter, cyclin A2 promoter, Cdc2 promoter, Cdc25C promoter, cyclin Epromoter, Cdc6 promoter, DHFR promoter and histones promoters. 6: Theconstruct of claim 4, wherein said IRES is selected from the groupconsisting of G2-IRES, HCV IRES, ODC IRES, c-myc IRES and p58 PITSLREIRES. 7: The construct of claim 1, wherein said destruction controlelement is selected from the group consisting of cyclin B1 D-box, cyclinA N-terminus, KEN box, cyclin E and p27Kip1. 8: The construct of claim1, wherein said live-cell detectable reporter molecule is operablylinked to and under the control of a cell cycle phase-specific spatiallocalisation control element. 9: The construct of claim 8, wherein saidcell cycle phase-specific spatial localisation control element is thecyclin B1 cytoplasmic retention sequence (CRS) including its NES. 10:The construct of claim 1, wherein said live-cell reporter molecule isselected from the group consisting of fluorescent proteins and enzymes.11: The construct of claim 10, wherein said fluorescent protein isselected from Green Fluorescent Protein (GFP) and a functional GFPanalogue in which the amino acid sequence of wild type GFP has beenaltered by amino acid deletion, addition, or substitution. 12: Theconstruct of claim 10, wherein said enzyme reporter is selected from thegroup consisting of a β-lactamase and nitroreductase. 13: The constructof claim 1, including a cyclin B1 promoter, a cyclin B1 destruction box(D-box), a cyclin B1 cytoplasmic retention sequence (CRS) and a greenfluorescent protein (GFP). 14: A nucleic acid reporter constructincluding an expression vector comprising: a) a vector backbonecomprising: i) a bacterial origin of replication; and ii) a bacterialdrug resistance gene; b) a cell cycle phase specific expression controlelement; c) a destruction control element; and d) a nucleic acidsequence encoding a reporter molecule. 15: The construct of claim 14,further comprising a cell cycle phase-specific spatial localisationcontrol element and/or a eukaryotic drug resistance gene. 16: A hostcell transfected with the construct of claim
 1. 17-18: (cancelled). 19:A method for determining the cell cycle position of a mammalian cell,comprising: a) expressing in cell the nucleic acid reporter construct ofclaim 1; and b) determining the cell cycle position by monitoringsignals emitted by the reporter molecule. 20: A method of determiningthe effect of a test system on the cell cycle position of a mammaliancell comprising: a) expressing in said cell in the absence and in thepresence of said test system a nucleic acid reporter constructcomprising a nucleic acid sequence encoding a detectable live-cellreporter molecule operably linked to and under the control of: i) a cellcycle phase-specific expression control element, and ii) a destructioncontrol element; wherein said reporter construct is expressed in a cellat a predetermined point in the cell cycle; and b) determining the cellcycle position by monitoring signals emitted by the reporter moleculewherein a difference between the emitted signals measured in the absenceand in the presence of said test system is indicative of the effect ofthe test system on the cell cycle position of the cell. 21: A method ofdetermining the effect of a test system on the cell cycle position of amammalian cell comprising: a) expressing in said cell in the presence ofsaid test system a nucleic acid reporter construct comprising a nucleicacid sequence encoding a detectable live-cell reporter molecule operablylinked to and under the control of: i) a cell cycle phase-specificexpression control element, and ii) a destruction control element;wherein said reporter construct is expressed in a cell at apredetermined point in the cell cycle; and b) determining the cell cycleposition by monitoring signals emitted by the reporter molecule, c)comparing the emitted signal in the presence of the test system with aknown value for the emitted signal in the absence of the test system;wherein a difference between the emitted signal measured in the presenceof the test system and said known value in the absence of the testsystem is indicative of the effect of the test system on the cell cycleposition of the cell. 22: A method of determining the effect of a testsystem on the cell cycle position of a mammalian cell comprising: a)providing cells containing a nucleic acid reporter construct comprisinga nucleic acid sequence encoding a detectable live-cell reportermolecule operably linked to and under the control of: i) a cell cyclephase-specific expression control element, and ii) a destruction controlelement; wherein said reporter construct is expressed in a cell at apredetermined point in the cell cycle; b) culturing first and secondpopulations of said cells respectively in the presence and absence of atest system and under conditions permitting expression of the nucleicacid reporter construct; and c) measuring the signals emitted by thereporter molecule in said first and second cell populations; wherein adifference between the emitted signals measured in said first and secondcell populations is indicative of the effect of said test system on thecell cycle position of said cell. 23: A method of determining the effectof the mammalian cell cycle on the expression, translocation orsub-cellular distribution of a first detectable reporter which is knownto vary in response to a test system comprising: a) expressing in saidcell in the presence of said test system a second nucleic acid reporterconstruct comprising a nucleic acid sequence encoding a detectablelive-cell reporter molecule operably linked to and under the control of:i) a cell cycle phase-specific expression control element, and ii) adestruction control element; wherein said reporter construct isexpressed in a cell at a predetermined point in the cell cycle; b)determining the cell cycle position by monitoring signals emitted by thesecond reporter molecule; and c) monitoring the signals emitted by saidfirst detectable reporter; wherein the relationship between cell cycleposition determined by step b) and the signal emitted by the firstdetectable reporter is indicative of whether or not the expression,translocation or sub-cellular distribution of the first detectablereporter is cell cycle dependent. 24 (cancelled) 25: The method of claim20, wherein said test system is an agent selected from the groupconsisting of drugs, nucleic acids, hormones, proteins, and peptides towhich said cell is exposed. 26: The method of claim 20, wherein saidtest system is an agent selected from a peptide or protein that isexpressed in the cell under study. 27: A cell line comprising the cellof claim 16 for use in establishing xenografts in a host organism. 28: Acell line comprising the cell of claim 16 for use in establishingallografts in a host organism. 29: A transgenic organism comprising thecell of claim 16.